Methods of preparing pharmaceutical compositions for treatment of factor Vlll associated disorders and methods of use

ABSTRACT

The invention concerns glycosylated proteins having human factor VIII activity. In a preferred embodiment, the protein is glycosylated with oligosaccharides that include an alpha-(2,6)-linked sialic acid and a bisecting GlcNAc linked to a core beta-mannose.

This application is a divisional of U.S. application Ser. No.10/225,900, filed on Aug. 22, 2002, which is hereby incorporated byreference herein in its entirety, which is a continuation-in-part ofU.S. application Ser. No. 10/006,091, filed Dec. 6, 2001, now abandoned,which is a continuation of U.S. application Ser. No. 09/209,916, filedDec. 10, 1998, now U.S. Pat. No. 6,358,703, which is hereby incorporatedby reference herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field

The present invention relates to factor VIII glycoforms. In particular,this invention relates to a recombinantly produced factor VIII that hasa glycosylation pattern resembling the glycosylation pattern ofnaturally occurring human Factor VIII.

2. Background

Human factor VIII is a trace plasma glycoprotein involved as a cofactorin the activation of factor X and factor IXa. Inherited deficiency offactor VIII results in the X-linked bleeding disorder hemophilia A,which can be treated successfully with purified factor VIII. Thereplacement therapy of hemophilia A has evolved from the use ofplasma-derived factor VIII to the use of recombinant factor VIIIobtained by cloning and expressing the factor VIII cDNA in mammaliancells. (Wood et al., 1984, Nature 312: 330).

Human factor VIII has a polypeptide molecular weight of 265,000. FactorVIII has three types of domains. It has a domain organization ofA1-A2-B-A3-C1-C2 and is synthesized as a single chain polypeptide of2351 amino acids, from which a 19-amino acid signal peptide is cleavedupon translocation into the lumen of the endoplasmic reticulum. The Bdomain contains up to 50% of the mass of the factor VIII and has noknown function. Due to proteolysis within the B domain and between theA2 and B domains, plasma-derived and recombinant factor VIII areisolated as a heterogeneous population of heterodimers with little or nosingle chain factor VIII present. It is likely that factor VIIIcirculates in predominantly heterodimeric form. (Lollar, Peter, 1995,Inhibitors to Coagulation Factors, edited by Louis Aledort et al.,Plenum Press, pp. 3-17).

Factor VIII is also described as consisting of three major regions: anN-terminal 90-kd heavy chain, a C-terminal 80-kd light chain, and thecentral B domain.

Factor VIII is heavily glycosylated. Glycosylation involves themodification of the polypeptide backbone with one or moreoligosaccharide groups. Glycosylation can dramatically affect thephysical properties of proteins and can also be important in proteinstability, secretion, and subcellular localization. Proper glycosylationcan be essential for a protein's biological activity. For example, thecirculation half-life of recombinant erythropoietin, a hormone involvedin the regulation of the level of red blood cells, was greatly increasedwhen its glycosylation pattern was changed. For years Amgen discarded80% of the recombinant erythropoietin it generated because of inadequateglycosylation, which resulted in unacceptably rapid clearing from theblood. When two extra sugars were added to those normally found onerythropoietin, a new drug, sold as ARANESP® erythropoiesis stimulatingprotein, was developed that stays in the blood much longer than theoriginal drug and thus requires less frequent dosing. (Maeder, Thomas,Sci. Amer., July 2002, pp. 40-47).

The oligosaccharide groups that create mammalian glycosylation patternsare derived from roughly ten simple sugars that can join with each otherat many different points to form intricate branching patterns. Not onlycan a sugar add to another sugar at many different locations in thefirst sugar's structure, but the addition can also be in differentorientations such as when the newly added sugar points above or belowthe plane of the ring of the first sugar. Because of these two factors,even the simplest sugars in the human body can combine in so manydifferent ways that more than 15 million four-component oligosaccharidesare theoretically possible. (Id.).

Glycosylation occurs at specific locations on the polypeptide backbone.It occurs typically when O-linked oligosaccharides are attached tothreonine or serine residues and when N-linked oligosaccharides areattached to asparagine residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. Althoughdifferent oligosaccharides are present in glycosylation, one sugar,N-acetylneuraminic acid (commonly known as sialic acid), is commonlyfound on both N-linked and O-linked oligosaccharides. Sialic acid isusually the terminal sugar residue on N-linked and O-linkedoligosaccharides.

Human factor VIII has 25 potential N-linked glycosylation sites, 19 ofwhich are in the B domain. Of the glycosylation sites in the B domain,at least 75% are occupied. The A1 subunit has two potential N-linkedglycosylation sites, at least one of which is occupied. The A2 subunithas a single unoccupied site. The light chain (subunits A3, C1 and C2)has two potential N-linked glycosylation sites, at least one of which isoccupied. (Lollar, supra, at 1-5).

Due to the fact that factor VIII is heavily glycosylated, high-levelexpression (>0.2 pg/c/d) of recombinant factor VIII has been difficultto achieve (Lind et al., 1995, Eur J Biochem. 232: 19-27; Kaufman etal., 1989, Mol Cell Biol. 9: 1233-1242). Expression of factor VIII inmammalian cells is typically 2-3 orders of magnitude lower than thatobserved with other genes using similar vectors and approaches. Theproductivity of production cell lines for factor VIII has been in therange of 0.5-1 μU/c/d (0.1-0.2 pg/c/d).

It has been demonstrated that the B-domain of factor VIII is dispensablefor procoagulant activity. Because the majority of the glycosylationsites are in the B domain, the overall size of the full-length factorVIII molecule is greatly decreased by deleting this domain. Usingtruncated variants of factor VIII, improved expression of factor VIII inmammalian cells has been reported by various groups (Lind et al., 1995,Eur J Biochem. 232: 19-27; Tajima et al., 1990, Proc 6^(th) Int SympH.T. p. 51-63; U.S. Pat. No. 5,661,008 to Almstedt, 1997). However, theexpression level of the factor VIII variants remained below 1 pg/c/dfrom a stable cell clone.

Variants of B-domain deleted recombinant Factor VIII have been made. Forexample, one variant, referred to herein as BDD FVIII SQ (SEQ ID NO: 1),has been genetically engineered to replace the 908 amino acids of the Bdomain with a short 14 amino acid linker that is derived from the N- andC-terminal ends of the B domain. BDD FVIII SQ is sold by Wyeth/GeneticsInstitute as REFACTO® anti-hemophilic factor. It is produced in Chinesehamster ovary (CHO) cells and secreted as a heterodimer. (Sandberg, H.et al., 2001, Seminars in Hematology, Vol. 38, No. 2, Suppl. 4, pp.4-12).

BDD FVIII SQ contains six consensus N-linked glycosylation sites. Threesites are in the heavy chain at Asn⁴¹, Asn²³⁹ and Asn⁵⁸² while threesites are in the light chain at Asn¹⁶⁸⁵, Asn¹⁸¹⁰ and Asn²¹¹⁸. In BDDFVIII SQ produced in CHO cells, four of the six N-linked consensus sitesare glycosylated and no N-linked carbohydrate was detected at theremaining consensus sites. Glycosylation was noted at Asn⁴¹, Asn²³⁹,Asn¹⁸¹⁰ and Asn²¹¹⁸. Most of the glycans attached to Asn239 and Asn²¹¹⁸are high-mannose structures, while the majority of the glycans at Asn⁴¹and Asn¹⁸¹⁰ are of the complex type and were predominantly sialyated,core fucosylated, and bi- and tri-antennal glycans withpoly-N-acetyllactosamine repeat units. (Id. at 8).

It is an object of the present invention to provide a recombinantlyproduced molecule having factor VIII activity for use as a humanpharmaceutical that can be produced in high yield. It is believed thatthe biological efficacy of such a molecule would be enhanced by having aglycosylation pattern, including specific oligosaccharide structures,that resembles or is identical to the glycosylation pattern in naturallyproduced human factor VIII. In particular, it is believed that the invivo half-life in humans of a protein having factor VIII activity wouldbe increased if the protein had a human glycosylation pattern. Also, itis believed that such a protein may have a higher specific activity invivo. Therefore, it is an object of the present invention to provide amolecule having factor VIII activity that also has one or moreoligosaccharides attached at N-linked glycosylation sites that areidentical to or closely resemble the oligosaccharides found at N-linkedglycosylation sites in naturally produced human factor VIII.

SUMMARY OF THE INVENTION

We have now discovered an isolated glycosylated protein having factorVIII procoagulant activity and a human glycosylation pattern that can beproduced in high yield. More particularly, the glycosylation patternincludes an N-linked oligosaccharide that contains alpha-(2,6)-linkedsialic acid and a bisecting N-acetylglucosamine (GlcNAc) linked to acore beta-mannose. Because such a glycosylation pattern is present innaturally occurring human factor VIII, it is believed that thepharmaceutical properties of the inventive protein are superior to otherisolated proteins having factor VIII activity but not an N-linkedoligosaccharide that contains alpha-(2,6)-linked sialic acid and/or abisecting N-acetylglucosamine (GlcNAc) linked to a core beta-mannose.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino Acid Sequence of BDD FVIII SQ (SEQ ID NO:1).

FIG. 2. Sequence of terminal repeat (TR) sequence isolated fromEpstein-Barr virus (SEQ ID NO:2).

FIG. 3. Plasmid map of pCIS25DTR.

FIG. 4( a). Derivation of clone 20B8.

FIG. 4( b). Comparison of productivities of several clones in variousmedia. Three data points are presented from a two month stability testof each clone.

FIG. 5. Volumetric productivity of clone 20B8.

FIG. 6. Anion exchange chromatographic profile of 2-aminobenzamidelabeled oligosaccharides from BDD FVIII SQ produced in HKB11 cells. Thetop panel represents total oligosaccharide (OS) pool, the middle panelrepresents total OS pool digested with NDV neuraminidase and the bottompanel represents total OS pool digested with A. ureafaciensneuraminidase.

FIG. 7. MALDI mass spectrometric analysis of BDD FVIII SQ produced inHKB11 cells.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, isolated glycosylated proteinshaving factor VIII activity are provided. In a preferred embodiment, theprotein is glycosylated with oligosaccharides that include analpha-(2,6)-linked sialic acid and/or a bisecting GlcNAc linked to acore beta-mannose.

Preferably, the invention is directed to a glycosylated proteincomprising the amino acid sequence of SEQ ID NO:1 and a humanglycosylation pattern. More preferably, the invention is directed to aglycosylated protein comprising the amino acid sequence of SEQ ID NO:1and oligosaccharide comprising alpha-(2,6)-linked sialic acid and/or abisecting GlcNAc linked to a core beta-mannose. Preferably, thisglycosylated protein is isolated.

In another embodiment, the invention is directed to a glycosylatedprotein having a 90-kd heavy chain and an 80-kd light chain linked by alinker polypeptide of about 14 amino acids, wherein the protein hasfactor VIII procoagulant activity in humans and a human glycosylationpattern. Preferably, the invention is directed to a glycosylated proteinhaving a 90-kd heavy chain and an 80-kd light chain linked by a linkerpolypeptide of about 14 amino acids, wherein the protein has factor VIIIprocoagulant activity in humans and N-linked oligosaccharides comprisingalpha-(2,6)-linked sialic acid and/or a bisecting GlcNAc linked to acore beta-mannose.

In another embodiment, the invention is directed to a protein havingFactor VIII procoagulent activity, wherein the amino acid sequence ofthe protein and the amino acid sequence of SEQ ID NO:1 have at least 62%identity and the protein has an N-linked oligosaccharide comprisingalpha-(2,6)-linked sialic acid and/or a bisecting GlcNAc linked to acore beta-mannose. More preferably, the percent identity is at least72%, still more preferably at least 82%, yet more preferably at least92%, and still yet more preferably at least 95%.

Percent identity is determined from an optimal global alignment betweenthe two sequences being compared. An optimal global alignment isachieved using, for example, the Needleman-Wunsch algorithm disclosed atNeedleman and Wunsch, 1970, J. Mol. Biol. 48:443-453, which is herebyincorporated herein in its entirety. Preferably, percent identity isdetermined by using the Needle implementation of the Needleman-Wunschalgorithm, which is available at the website of the EuropeanBioinformatics Institute, EMBL-EBI, www.ebi.ac.uk. “Identity” means thatan amino acid at a particular position in a first polypeptide isidentical to a corresponding amino acid in a second polypeptide that isin an optimal global alignment with the first polypeptide. By thestatement “sequence A is n % identical to sequence B” is meant that n %of the positions of an optimal global alignment between sequences A andB consists of identical residues. Optimal global alignments in thisdisclosure used the following parameters in the Needleman-Wunschalignment algorithm for polypeptides: Substitution matrix: blosum62. Gapscoring function: Gap penalty 10.0, Extend penalty 0.5.

The invention is also directed to pharmaceutical compositions comprisinga therapeutically effective amount of one or more of the glycosylatedproteins of the invention in admixture with a pharmaceuticallyacceptable adjuvant. Glycosylated proteins having Factor VIII activityare preferably administered parenterally. Preferred formulations forparenteral administration include buffered saline and albumin. Alsopreferred are the formulations disclosed in U.S. Pat. No. 5,763,401,issued Jun. 9, 1998 (Rajiv Nayar), which is hereby incorporated byreference herein in its entirety. Another preferred formulation includessodium chloride, sucrose, L-histidine, calcium chloride and polysorbate80. Another preferred formulation includes sucrose, glycine, histidine,calcium chloride, sodium chloride, polysorbate 80, imidazole,tri-n-butyl phosphate and copper. Preferred formulations do not includeany toxic agents, such as toxic solubilizing agents like sodium dodecylsulfate (SDS).

The required dosages are within the skill of those in the art todetermine. For example, minor hemorrhaging in a patient with severehemophilia A may be treated with 10-20 international units (IU) per kgbody weight, which can be repeated if evidence of further bleeding.Moderate to major hemorrhaging in such a patient may be treated with15-30 IU per kg body weight, which can be repeated at 12-24 hours ifneeded. Major to life-threatening hemorrhaging in such a patient may betreated with an initial dose of 40-50 IU/kg repeated at a dose of 20-25IU/kg every 8 to 12 hours. For major surgical procedures to such apatient, the protein can be administered preoperatively at 50 IU/kgrepeated as necessary after 6 to 12 hours. One IU, as defined by theWorld Health Organization standard for blood coagulation Factor VIII,human, is approximately equal to the level of Factor VIII activity foundin 1 mL of fresh pooled human plasma.

The invention is also directed to methods of treating a factorVIII-associated disorder such as hemophilia by administering to a humanin need thereof a therapeutically effective amount of the pharmaceuticalcompositions of the invention. The invention is also directed to methodsof preparing one of the pharmaceutical compositions of the invention bymixing the protein having factor VIII procoagulent activity with apharmaceutically acceptable adjuvant.

Preferably, the glycosylated protein is the product of recombinant cellproduction, and the glycosylation is the result of the normalpost-translational cell functioning of the host cell. For example, thevector and cell line described in Example 1 infra can be used to producethe glycosylated protein of the invention.

Alternatively, glycosylation can be achieved through chemicalmodification of a protein having factor VIII activity. In yet a furtheralternative, the protein can be glycosylated by the addition of anenzyme that acts to add alpha-(2,6)-linked sialic acid to a host cellthat expresses the protein having factor VIII activity but does notendogenously produce such an enzyme. For example, dihydrofolatereductase (dhfr) deficient CHO cells are commonly used host cells forrecombinant glycoprotein production. Yet CHO cells do not endogenouslyexpress the enzyme beta-galactoside alpha-2,6 sialyltransferase, whichis used to add sialic acid in the 2,6 linkage to galactose on themannose alpha-1,3 branch. To add sialic acid at this linkage to aprotein produced in CHO cells, the CHO cells can be transfected with afunctional beta-galactosidase alpha-2,6 sialyltransferase gene to allowfor incorporation of sialic acid in the 2,6 linkage to galactose asdesired. (See Lee et al., J. Biol. Chem., 1989, 264:13848 for discussionof techniques for creating modified CHO cells).

Similarly, a bisecting GlcNAc can be added to a recombinantly producedprotein having factor VIII activity by transfecting a host cell thatdoes not endogenously produce this oligosaccharide linkage with thefunctional gene for the enzyme N-acetylglucosaminyltransferase, whichhas been reported to catalyze formation of a bisecting GlcNAc structure.

The proteins of the present invention can be produced at the industrialscale using the newly created cell host described in Example 1 atspecific productivities in the range of 2-4 pg/cell/day (10-20 μU/c/d).Under serum-free conditions, one clone has sustained a dailyproductivity of 2-4 pg/c/d. Clones with this high level of productivityare able to produce 3-4 million units per day in a 15-liter perfusionfermenter. One unit of factor VIII activity is by definition theactivity present in one milliliter of plasma. One pg of factor VIII isgenerally equivalent to about 5 μU of factor VIII activity.

As used herein, a protein having factor VIII procoagulant activity is aprotein that causes the activation of factor X in an in vitro or in vivomodel system. As non-limiting examples, this definition includesfull-length recombinant human factor VIII and BDD FVIII SQ (SEQ IDNO: 1) whose sequence is described in FIG. 1.

As used herein, a human glycosylation pattern in a protein having factorVIII activity is a pattern of O- or N-linked oligosaccharides that arefound in naturally occurring human factor VIII, when only theoligosaccharides in the domains or attached to the N- and O-linkedglycosylation sites that are shared in common between the protein andnaturally occurring factor VIII are compared. For example, in arecombinant B domain-deleted factor VIII, a human glycosylation patternis found when the same N- or O-linked oligosaccharides found innaturally occurring human factor VIII (excepting those in the B domain)are found in the recombinant B domain-deleted factor VIII.

It is understood that the inventive glycosylated proteins may containother oligosaccharides in addition to the two specified oligosaccharidesdiscussed herein, namely, alpha-(2,6)-linked sialic acid and/or abisecting GlcNAc linked to a core beta-mannose.

As used herein, an isolated protein is a protein substantially free ofother proteins. For example, isolated proteins of the invention are atleast 50%, more preferably at least 75% and still more preferably atleast 90% by weight of the total protein matter present. An isolatedprotein having Factor VIII procoagulent activity preferably has anactivity of greater than 5000 IU/mg protein, and more preferably has anactivity of greater than 10,000 IU/mg protein.

In the case of amino acid sequences that are less than 100% identical toa reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Proteins referred to herein as “recombinant” are proteins orpolypeptides produced by the expression of recombinant nucleic acids.

A high level of expression of a protein having factor VIII procoagulantactivity means at least about 2 μU/c/d, or more preferably at leastabout 4 μU/c/d, or most preferably at least about 5 μU/c/d, of factorVIII activity if grown in plasma derived protein-free medium, or atleast about 4 μU/c/d, or more preferably at least about 8 μU/c/d, ormost preferably at least about 10 μU/c/d, of factor VIII activity ifgrown in medium supplemented with plasma derived protein. When theprotein expressed is BDD-FVIII, cell lines having specificproductivities up to about 15 μU/c/d, more preferably up to about 20μU/c/d may be obtained by the method described herein.

As used herein to describe the origin of cell lines, “derived from” isintended to include, but not be limited to, normal mitotic cell divisionand processes such as transfections, cell fusions, or other geneticengineering techniques used to alter cells or produce cells with newproperties.

EXAMPLES Example 1 Preparation of BDD FVIII SQ 1.1 FVIII Assay

The activity of factor VIII derivatives obtained from recombinant geneexpression in methotrexate (MTX)-resistant cell populations was measuredby a chromogenic assay. Activity was quantitated using COATEST® factorVIII:C/4 kit, a diagnostic test kit for measuring factor VIII:C activityusing a chromogenic substrate assay, (Cromogenix, Molndal, Sweden)according to manufacturer's instructions. A U.S. standardanti-hemophilic factor (factor VIII) known as MEGA 1 (Office ofBiologics Research and Review, Bethesda, Md.) was used as the standardof measurement in this assay (see Barrowcliffe, 1993, Thromb Haem 70:876).

1.2 Construction of Expression Vectors for B-Domain Deleted FVIII

The sequence of the BDD FVIII SQ is shown in FIG. 1. The 90-kD and 80-kDchains were linked by a linker consisting of 14 amino acids. (See U.S.Pat. No. 5,952,198, issued Sep. 14, 1999 (Sham-Yuen Chan) for productionmethod, which is hereby incorporated by reference herein in itsentirety). The expression vector for BDD FVIII SQ was made usingstandard recombinant DNA techniques. The structure of the expressionvector (pCIS25DTR) is shown in FIG. 3. The vector includes atranscriptional unit for BDD FVIII SQ and a selectable marker,dihydrofolate reductase (dhfr). In addition, a terminal repeat sequencefrom Epstein-Barr virus, which shows enhanced drug selection ratio,(FIG. 2) was inserted into the vector to increase the integrationefficiency. The vector is essentially a construct of a vector (depositedATCC 98879) that has been engineered to include a transcriptional unitcorresponding to the sequence shown in FIG. 1. (See U.S. Pat. No.6,180,108, issued Jan. 30, 2001 (Myung-Sam Cho and Sham-Yuen Chan) fordiscussion of the terminal repeat sequence, which is hereby incorporatedherein in its entirety).

Similar vectors can be constructed and used by those having skill in theart to obtain cells expressing proteins having factor VIII procoagulantactivity. For example, coding sequences coding for known variants offactor VIII which retain procoagulant activity can be substituted forthe BDD FVIII SQ coding sequence. Also, instead of dhfr, otherselectable markers can be used, such as glutamine synthetase (gs) ormultidrug-resistance gene (mdr). The choice of a selection agent must bemade accordingly, as is known in the art, i.e. for dhfr, the preferredselection agent is methotrexate, for gs the preferred selection agent ismethionine sulfoximine, and for mdr the preferred selection agent iscolchicine.

1.3 Derivation of Cell Lines Expressing BDD FVIII SQ: Transfection, DrugSelection and Gene Amplification

Thirty micrograms of pCIS25DTR DNA was transferred into HKB11, which isa hybrid of 293S cells and human Burkitt's lymphoma cells. (See U.S.Pat. No. 6,136,599, issued Oct. 24, 2000 (Myung-Sam Cho), incorporatedherein by reference in its entirety). Samples of HKB11 were deposited atthe American Type Culture Collection (ATCC, 10801 University Blvd.,Manassas, Va. 20110-2209) (ATCC deposit no. CRL-12568) on Sep. 16, 1998.The DNA was transferred into the cells by electroporation set at 300volts and 300 micro farads (BTX Electro cell Manipulator 600) using a 2mm cuvette (BTX part #620). In comparative experiments done to parallelwork with the HKB11 cells, CHO (Chinese hamster ovary) and 293S (humanembryonic kidney) cells were transfected using a cationic lipid reagentDMRIE-C (Life Technologies, Gaithersburg, Md.) according to a protocolprovided by the Life Technologies. Amplification of transfected cellswas done with increasing methotrexate (MTX) concentrations (100 nM, 200nM, 400 nM, and 800 nM) at 1×10⁶ cells per 96 well plate in aMTX-selection medium lacking hypoxanthine and thymidine (DME/F12 mediawithout hypoxanthine and thymidine plus 5% dialyzed fetal bovine serumfrom Hyclone, Logan, Utah). MTX resistant cells were scored for growth,and secretion of the BDD-FVIII was screened using a COATEST® factor VIIIchromogenic substrate assay kit about 2-3 weeks post-transfection. Thecultivation of cells were done at 37° C. in a humidified 5% CO₂incubator.

1.4 Limiting Dilution Cloning

Single cell clones (SCC) were derived by limiting dilution cloning (LDC)of high producing populations in 96 well plates under serum-freeconditions. Cells were seeded at 1-10 cells per well in DME/F12 mediasupplemented with HUMULIN® recombinant insulin (Lilly, Indianapolis,Ind.) at 10 μg/ml, 10× essential amino acids (Life Technology,Gaithersburg, Md.), and PLASMANATE® human plasma protein fraction(Bayer, Clayton, N.C.). PLASMANATE® human plasma protein (HPP) fractioncontains human albumin (88%) and various globulins (12%). The cloneswere screened for BDD-FVIII productivity using the COATEST® factor VIIIchromogenic substrate assay kits. The highest producing clones wereselected for stability evaluation in shake flasks. For HKB cells, thefirst round LDC was performed using selection medium supplemented with5% dialyzed FBS. The second round LDC was done in serum-free butPLASMANATE® HPP fraction-containing medium using the first SCC adaptedin serum-free medium supplemented with PLASMANATE® HPP fraction.

1.5 Derivation of HKB Clone 20B8

As summarized in FIG. 4( a), the initial population IC10 was derivedfrom the HKB cells transfected with pCIS25DTR after amplification with400 nM MTX in the selection medium with 5% FBS. One of the first singlecell clones (SCCs), 10A8, derived from 1C10 by a LDC using a selectionmedium supplemented with 5% FBS was adapted in serum-free mediumsupplemented with PLASMANATE® HPP fraction. Unexpectedly, 10A8 showedextremely increased levels of rFVIII production at this stage (FIG. 4b). Therefore, we did a second LDC using the medium supplemented withPLASMANATE® HPP fraction. The productivity of SCCs (e.g. 20B8) derivedfrom the second LDC was similar with PLASMANATE® HPP fraction-adapted10A8. 20B8 showed higher levels of BDD-FVIII than original 10A8 derivedfrom the first LDC in serum-containing medium. Finally, 20B8 was adaptedto growth in plasma protein-free (PPF) medium. Samples of 20B8 weredeposited at the American Type Culture Collection (ATCC, 10801University Blvd., Manassas, Va. 20110-2209) (ATCC deposit no. CRL-12582)on Oct. 6, 1998.

As shown in Table 1, HKB clones exhibit superior productivity forBDD-FVIII. A 10-20 fold increase in productivity was observed in HKBcells when compared to clones derived from transfected CHO and 293Scells. HKB cells, which do not form large aggregates of cells when grownin suspension culture, are preferred cells for the expression ofproteins having factor VIII procoagulant activity.

TABLE 1 Expression of FVIII and BDD FVIII SQ in human and rodent celllines Specific Productivity (μU/c/d)* FVIII Derivatives BHK 293s CHO HKBFull length FVIII 0.45 1.2 0.5 1.0 BDD FVIII SQ ND 2.5 1.0 20 *Averageof 5 high producing clones (in serum-free media) ND = Not done

1.6 Plasma-Protein-Free Adaptation of Clones

HKB clones that have been adapted to grow as serum-free suspensioncultures were further weaned of plasma protein supplements. The weaningwas done in sterile polycarbonate shake flasks (Corning, Corning, N.Y.)at a cell density of about 0.5×10⁶ cells/ml using plasma derived proteinfree medium. The plasma protein free (PPF) medium was DME/F12 mediumsupplemented with pluronic F68 (0.1%), CuSO₄ (50 nM), and FeSO₄/EDTA (50μM). Complete medium exchange was done every 48 hours and the shakeflasks were re-seeded at 0.5×10⁶ cells/ml.

1.7 Fermentation of Clone 20B8

The productivity of clone 20B8 was evaluated in a 15-liter perfusionfermenter. The fermenter was seeded with clone 20B8 cells at a densityof about 3×10⁶ cells/ml. The fermenter was perfused at a rate of 4volumes per day with the serum-free production medium as described inthe preceding paragraph. A final cell density of 2×10⁷ cells/ml wassustained throughout the evaluation period (45 days). As shown in FIG.5, during the first 4 weeks of fermentation, clone 20B8 was perfusedwith the serumfree production medium supplemented with PLASMANATE® HPPfraction and was able to sustain high productivity. From day 28 to theend of the fermentation run, the cells were perfused with the sameserumfree production medium but without PLASMANATE® HPP fraction. Asshown in FIG. 5, the cells continued to produce high levels of FVIII ina plasma derived protein-free environment. “Plasma derived protein-free”means that essentially no proteins isolated from plasma have been addedto the medium.

Example 2 Characterization of Type of Linkage of Sialic Acid of BDDFVIII SQ

BDD Factor VIII SQ was purified as described in Biochemistry25:8343-8347 (1986) using ion exchange and affinity chromatgraphy. Thematrix 2,5-dihydroxybenzoic acid (DHB) was purchased from AldrichChemical Company, USA. HPLC grade trifluoroacetic acid (TFA) was fromPierce, USA. Baker analyzed HPLC grade acetonitrile was from J T Baker,USA. Newcastle disease virus neuraminidase was purchased from SigmaChemical Co., USA. All consumable reagents for the GlycoPrep 1000,2-amino benzamide (2-AB), GlycoSep C column, A. ureafaciensneuraminidase and standard oligosaccharides were purchased from OxfordGlycoSciences (OGS), Abingdon, UK.

Oligosaccharide analyses were done by dialyzing purified BDD FVIII SQagainst Milli-Q water to remove salt and buffers. The desalted BDD FVIIISQ was dried in glass reactor vials for 18 hours using a SpeedVac.Oligosaccharides were released by chemical hydrazinolysis using aGlycoPrep 1000 system from OGS. The liberated pool of oligosaccharideswas filtered and dried immediately in a SpeedVac to minimize the loss ofterminal sialic acids.

The released total oligosaccharide pool was coupled to 2-aminobenzamide(2-AB). Briefly, oligosaccharides were dissolved in 5 ml of a solutionof 2-AB (0.35M) in dimethylsulfoxide/glacial acetic acid (30% v/v)containing sodium cyanoborohydride (1 M). The glycan solution was thenincubated at 65° C. for 2 h. After the conjugation with 2-AB, thereaction mixture was applied to a cellulose disk (1 cm in diameter) in aglass holder. The disk was washed with 1 ml of acetonitrile followed by5×1 ml 4% deionized water in acetonitrile to remove unreacted dye andnon-glycan materials. Labeled glycans were eluted using three washes(0.5 ml) of water and then filtered (0.2 mm).

Labeled oligosaccharides were separated with a DEAE anion-exchangecolumn on the basis of their terminal sialic acid content. The columnsize was 4.6 mm×100 mm with a bed volume of 1.7 ml. A solvent gradientsystem of 0-200 mM ammonium acetate in 20% acetonitrile for 40 min at0.3 ml/min was used. A high performance liquid chromatographic systemequipped with an HP Ti-series 1050 pump (Hewlett Packard) was used todeliver the solvents and a programmable fluorescence detector (HewlettPackard, model 1046A, λ_(exc)=330 nm and λ_(emiss)=420 nm) was used todetect 2-amino benzamide labeled oligosaccharide peaks.

Oligosaccharide pools were desialylated by digesting with eitherArthrobacter ureafaciens (Sigma, cat no N-3642) or Newcastle diseasevirus (OGS, cat no. X-5017) neuraminidase in 50 mM sodium acetatebuffer, pH 5.0 for 6 h or 18 h at 37° C. Digested samples were purifiedon a microcolumn containing 150 μL each of DOWEX® AG50, CHELEX® 100,DOWEX® AG3 and QAE SEPHADEX® column chromatography resins. Samples wereeluted with water, then rotary-evaporated to dryness before analysis.

FIG. 6 shows the anion-exachange chromatographic profile of2-aminobenzamide labeled oligosaccharides from BDD FVIII SQ As shown inthe middle panel of FIG. 6, digestion of BDD FVIII SQ with neuraminidasefrom Newcastle disease virus that is specific for alpha (2,3)-linkedsialic acid did not eliminate all the sialic acid peaks. Upon digestionof BDD FVIII SQ with neuraminidase from A. ureafaciens (capable ofremoving sialic acid of alpha (2,3) and alpha-(2,6) linkages), all thesialic acid peaks were eliminated (bottom panel of FIG. 6) as comparedto undigested BDD FVIII SQ (top panel of FIG. 6). The neuraminidasedigestion studies indicate that BDD FVIII SQ is capped with sialic acidof both alpha-(2,3) and alpha-(2,6) linkage.

Example 3 Characterization of Bisecting Oligosaccharide Linkage of BDDFVIII SQ Using MALDI Mass Spectrometric Analysis

The 2,5-dihydroxybenzoic acid (DHB) matrix was prepared by dissolving 10mg DHB in 1 mL 70% acetonitrile. Sample plates were dried after loadingsample and matrix.

The mass spectrometer used to acquire the spectra was a Voyager DE-RP(PerSeptive Biosystems, Inc., Framingham, Mass.). All samples (proteinand carbohydrates) were analyzed using delayed extraction mode andwithout the reflectron by irradiating with UV light (337 nm) from a N₂laser. Neutral oligosaccharides were analyzed in the positive-ion modeat 25 kV using the DHB matrix. The delay time was set at 100 ns foroligosaccharide samples and at 150 ns for protein samples. The gridvoltage was set to 94% and 89.5% of the accelerating voltage foroligosaccharides.

A two point external calibration (oligosaccharides high-mannose Man-5[(M+Na)⁻ _(Avg)1258] and NA3 [desialylated triantennary, (M+Na)⁻ _(Avg)2029]) was used for mass assignment of the ions. Typically, spectra from128-264 laser shots were summed to obtain the final spectrum.

As shown in FIG. 7 and Table 2, the FVIII-SQ has complex carbohydratestructures. These structures consist of biantennary, tri- andtetra-antennary oligosaccharide alditols. In addition, a uniquebisecting GlcNAc was identified. This bisecting GlcNAc structure hasbeen reported to be catalyzed by N-aceylglucosaminyltransferase, anenzyme known to be expressed in human cells.

TABLE 2 Major sodium ion (Na+)-associated oligosaccharide structures inHKB11-produced BDD FVIII SQ. Oligosaccharide Structures Theoretical massObserved mass detected in HKB-rFVIII (with Na+ ion) (with Na+ ion)M₃Gn₂G₂ (Biantennary) 1664 1667 M₃Gn₂G₁Gn_(b) (Bisecting) 1705 1708M₃Gn₂G₂F (Biantennary + 1810 1812 Fucosyl) M₃Gn₃G₃ (Triantennary) 20312033 n.d. ? 2179 M₃Gn₄G₄ (Tetraantennary) 2392 2391 n.d. Not-identifiedGn = N-acetylglucosamine; G = galactose; F = fucosyl; M = mannose

The above examples are intended to illustrate the invention and it isthought variations will occur to those skilled in the art. Accordingly,it is intended that the scope of the invention should be limited only bythe claims below.

1-24. (canceled)
 25. An isolated glycosylated protein having factor VIIIprocoagulant activity, wherein the amino acid sequence of the proteinhas at least 62% identity with SEQ ID NO:1 and the protein comprises anN-linked oligosaccharide having an alpha-(2,6)-linked sialic acid. 26.The isolated protein of claim 25, wherein the amino acid sequence has atleast 72% identity with SEQ ID NO:1.
 27. The isolated protein of claim25, wherein the amino acid sequence has at least 82% identity with SEQID NO:1.
 28. A pharmaceutical composition comprising the isolatedprotein of claim 25 and a pharmaceutically acceptable adjuvant.
 29. Thepharmaceutical composition of claim 28 wherein the composition issubstantially free of a toxic solubilizing agent.
 30. A method oftreating a factor VIII-associated disorder comprising administering to ahuman in need thereof a therapeutically effective amount of thepharmaceutical composition of claim
 28. 31. A method of reducinghemorrhage in a patient due to surgical procedure comprisingadministering to the patient a therapeutically effective amount of thepharmaceutical composition of claim
 28. 32. The method of claim 31,wherein the pharmaceutical composition is administered to the patientpreoperatively.
 33. The method of claim 32 further comprising the stepof re-administering the pharmaceutical composition 6 to 12 hours afterthe initial administration.
 34. An isolated glycosylated protein havingfactor VIII procoagulant activity, wherein the amino acid sequence ofthe protein has at least 62% identity with SEQ ID NO:1 and the proteincomprises an N-linked oligosaccharide having a bisecting GlcNAc linkedto a core beta-mannose.
 35. The isolated protein of claim 34, whereinthe amino acid sequence has at least 72% identity with SEQ ID NO:1. 36.The isolated protein of claim 34, wherein the amino acid sequence has atleast 82% identity with SEQ ID NO:1.
 37. A pharmaceutical compositioncomprising the isolated protein of claim 34 and a pharmaceuticallyacceptable adjuvant.
 38. The pharmaceutical composition of claim 37,wherein the composition is substantially free of a toxic solubilizingagent.
 39. A method of treating a factor VIII-associated disordercomprising administering to a human in need thereof a therapeuticallyeffective amount of the pharmaceutical composition of claim
 37. 40. Amethod of reducing hemorrhage in a patient due to surgical procedurecomprising administering to the patient a therapeutically effectiveamount of the pharmaceutical composition of claim
 37. 41. The method ofclaim 40, wherein the pharmaceutical composition is administered to thepatient preoperatively.
 42. The method of claim 41 further comprisingthe step of re-administering the pharmaceutical composition 6 to 12hours after the initial administration.